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PubMed1992

beta-Glycerophosphate-induced mineralization of osteoid does not alter expression of extracellular matrix components in fetal rat calvarial cell cultures.

When fetal rat calvarial cells are cultured in medium containing vitamin C, osteoid nodules develop after approximately 15 days of culture. Upon addition of an organic phosphate (beta-glycerophosphate, beta GP), these nodules mineralize. We have now used this system to explore the suggestion made by others that a negative feedback may exist between matrix mineralization on the one hand and the synthesis of alkaline phosphatase and bone matrix collagen on the other by analyzing the synthesis of these proteins and the levels of their mRNAs in mineralizing and nonmineralizing cultures. Our results indicate that in the osteoid nodule-bone nodule system, matrix mineralization did not affect the mRNA levels for osteopontin, type I collagen, bone sialoprotein, or osteocalcin. Synthesis of total protein and collagen and the osteocalcin content of culture media were also not different in the mineralizing and nonmineralizing cultures. However, alkaline phosphatase mRNA was increased in early mineralizing cultures and alkaline phosphatase activity in the cell layer was also increased in mineralizing cultures. Thus, the hypothesis that a direct negative feedback exists between mineralization and matrix protein synthesis is not supported by our experiments.

Alkaline Phosphatase↗
PubMed1999

Changes in the orientation of collagen fibers on the superficial layer of the mouse tibial bone after denervation: scanning electron microscopic observations.

This study was undertaken to evaluate the relationship between the mechanical stress loaded onto the bone and the orientation of collagen fibers formed by osteoblasts. The femoral, obturator, and sciatic nerves in the left posterior legs of 7-week-old mice were exposed and electroscissored to reduce the mechanical stress loaded onto the leg. Four weeks after operation, the tibial bones in the control and denervated legs were removed and observed by scanning electron microscopy (SEM) after NaOCl treatment. In the control right tibia, collagen fibers on the superficial bone matrix tended to be arranged parallel to the longitudinal axis of the bone. However, the arrangement of collagen fibers in the left tibia, which were immobilized for 4 weeks by denervation, was disorganized and ran in random directions. The findings suggest that the direction of collagen fibers in the bone changes in response to the mechanical stress loaded onto the bone, probably due to changes in the activity of osteoblasts in the denervated leg.

Animals↗
PubMed1998

Morphological and immunocytochemical characterization of primary osteogenic cell cultures derived from fetal rat cranial tissue.

Enzymatic digestion of bone tissue potentially releases a mixture of precursor, differentiating, and mature cells. Conceptually, early fetal osteogenic tissue should provide a more uniform population of cells than late embryonic or newborn bone in which cells have already differentiated. In this context, we have applied sequential enzymatic digestion to obtain and culture cells from 15-16-day fetal rat cranial tissue, a developmental age where deposition of bone matrix has not yet started at this site. These cultures were compared with those of osteogenic cells isolated from newborn rat calvariae and grown under similar conditions. Matrix production and composition were examined by colloidal gold immunocytochemistry using antibodies to bone sialoprotein (BSP), osteocalcin (OC), and osteopontin (OPN). The plated cells formed mineralized nodules by day 14. The presence of mineral was determined by von Kossa staining and backscattered electron imaging (BEI), and the accumulation of calcium and phosphorus within the nodules was demonstrated by X-ray microanalysis and elemental mapping. At early time intervals, cells were generally cuboidal in shape and showed a well-developed Golgi apparatus, which occasionally was immunoreactive for OPN. Labeling for BSP and OPN was found over mineralization foci and electron-dense material within, and at the periphery, of larger mineralized masses and over accumulations of afibrillar matrix at the dish surface. Osteocalcin immunoreactivity was also associated with electron-dense portions of the bone-like matrix. These data demonstrate the potential of presumptive fetal rat calvarial cells to form a bone-like matrix in vitro and suggest that the assembly and mineralization pattern show similarities to the process of intramembranous ossification. Such a culture system is of interest not only for studying cellular and matrix events of bone formation, but also factors which influence mesenchymal cells in committing themselves to the osteogenic pathway.

Animals↗
PubMed1986

The effects of immobilization on bone histomorphometry in rats.

To determine whether immobilization acts directly on bone by alteration of mechanical loading or systemically, studies of the effects of immobilization were carried out on histomorphometry of diaphyses of tibiae and on subcutaneous implants of demineralized allogenic bone matrix of rats. The right hind leg of growing rats was denervated by severing the tibial nerve. A sham operation on the right hind leg was performed in control animals. Bone formation at the endosteal and periosteal surfaces was significantly lower in tibiae from limbs with severed nerves as compared to tibiae from the intact limbs of nerve-sectioned rats and from both limbs of sham-operated control rats. Bone formation was decreased at both 3 and 7 weeks after immobilization. The decreased formation resulted in significant reductions in cross-sectional area. At 3 weeks post denervation, the periosteal bone formation rate was lower in tibiae of intact limbs from denervated rats as compared to tibiae from intact limbs of sham-operated animals. This finding was attributed to reduced physical activity of the denervated rats. In the implants, nerve section did not alter the amount of implant matrix resorbed, the amount of bone matrix synthesized, or the amount of calcium in the implant. These findings support the hypothesis that inhibition of bone formation at the tibial diaphysis in response to immobilization resulted from altered mechanical loading and not from the production of substances acting systemically. Whereas the mean medullary area of tibiae was not altered by nerve section, it was decreased in tibiae of all groups compared to the values of basal controls, indicating that bone formation was greater than bone resorption.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1984

The electric double layer in bone and its influence on stress-generated potentials.

Stress-generated potential (SGP) studies in fluid-filled bone have been interpreted in terms of streaming potential theory. Variations in fluid conductivity and viscosity are in agreement with this theory. Experimental results also indicate that the zeta potential of bone is dependent upon the ion concentration of the fluid. In fact, the zeta potential is seen to reverse polarity at sufficiently high concentrations of NaCl and KCl. This observation indicates that the electric double layer at the fluid-bone matrix interface is an important region contributing to electromechanical effects in bone. The double layer may also have an effect on piezoelectric studies in dry bone since the charged species remain on the matrix after the bone specimens are dried.

Animals↗
PubMed1992

Mineral-matrix interactions in bone and cartilage.

Mineral-matrix interactions regulate the process of hydroxyapatite formation in bones and teeth. In mineralizing tissues, many anionic macromolecules bind to mineral. By means of this binding, such molecules are able to regulate the size and shape of the mineral crystals, determine the site of initial crystal deposition, and determine the type of mineral crystals deposited. Collagen, which provides a template for hydroxyapatite deposition; extracellular matrix vesicles, which provide a protected environment for crystal deposition; and noncollagenous matrix proteins that have high affinities for hydroxyapatite have all been shown to affect mineralization in vitro. Some of the noncollagenous proteins have been shown to be capable of promoting and inhibiting mineral formation and growth, depending on their concentration and whether they are immobilized or free in solution. This review surveys the current understanding of mineral-matrix relationships involved in endochondral, intramembranous, and appositional bone formation, outlining the way in which mineral deposition is controlled in mammalian calcified tissues. The structural basis for the interaction of the matrix molecules with hydroxyapatite is presented, as is the in vitro and in situ data implicating the matrix molecules that interact with hydroxyapatite to control mineralization.

Bone Matrix↗
PubMed1996

Bone acidic glycoprotein-75 self-associates to form large macromolecular complexes.

Bone acidic glycoprotein-75 (BAG-75) displays a strong propensity to self-associate to form large fibrillar complexes above concentrations of 7 x 10(-8) M; acidic phosphoproteins osteopontin and bone sialoprotein do not form similar complexes. Although the majority of the data supporting this conclusion is derived from in vitro studies, the fact that similar sized complexes are observed in crude extracts of bone and calcified cartilage suggests that macromolecular BAG-75 complexes are also a component of mineralized matrices in vivo. An awareness of the existence of complexes in extracts from bone necessitates that these forms are accounted for in terms of the relative amounts of individual acidic phosphoproteins in bone matrix. We now estimate that the amount of BAG-75 in rat calvarial bone is equivalent to that of osteopontin. While BAG-75 is capable of binding up to 139 atoms of calcium/mole with an average affinity constant of 0.5-1.0 x 10(-3) M, millimolar concentrations of calcium are not required for self-association. Assuming macromolecular diffusion within osteoid is restricted, osteoblastic cells could control the extent of self-association through the rate at which BAG-75 is synthesized and secreted into the osteoid matrix. Based on these findings, we hypothesize that BAG-75 self-associates to form fibrillar complexes in vivo which function in a supportive mechanical role and/or as an electronegative ionic barrier. Electronegative BAG-75 barrier structures could play a role in concentrating phosphate ions within bone matrix, thus facilitating mineralization.

Animals↗
PubMed1995

Matrix proteins: potentials as body fluid markers of changes in the metabolism of cartilage and bone in arthritis.

Altered dynamics of cartilage and bone matrix in joint diseases results in increased release of macromolecules into synovial fluid (SF). Such macromolecules are increasingly explored as potential markers of damage and severity. This report focuses on the possibilities of using quantification of tissue specific markers for grading the tissue lesion at the molecular level in arthritis. The theoretical and experimental rationale for such an approach is supported by the results in clinical studies. These studies indicate that simultaneous analysis of multiple cartilage and bone markers in the same SF or serum sample could be useful for defining degree of tissue involvement in rheumatoid arthritis and, possibly, in osteoarthritis.

Arthritis, Rheumatoid↗
PubMed1990

[Experimental study of the mechanism of aluminum-induced bone disease].

The mechanism of aluminum-induced bone disease was studied by experiments in vitro and in rats. After decalcification femur sections were treated with 0.1 M AlCl3 and implanted with control bone matrices into weanling male rats. After 4 or 8 weeks of implantation the tissue values of calcium(Ca), phosphorus(P) and aluminum(Al) were measured. The results showed that both Ca and P values in Al-treated bones were obviously lower than those of the controls (P less than 0.01), Al level in these bones remained considerably high. Histological examination indicated that aluminum deposited diffusely in Al-treated bones, the bone formation rate and mineralization rate markedly decreased, osteoclastic and osteoblastic activities were also inhibited. In collagenase incubation test the bone matrices pretreated with different concentrations of AlCl3 showed different degrees of resistance to digestion of collagenase. It is considered that aluminum forms tight complexes with bone collagen causing cross linkage and destroying the inductive effect in bone matrix, thus leading to aluminum-induced bone disease. Our study also suggests that the usage of chelating agents can remove aluminum from matrix and recover its abilities of bone formation and mineralization.

Aluminum↗
PubMed1996

Identification of an osteocalcin gene promoter sequence that binds AP1.

Osteoblasts are differentiated cells that produce bone matrix components including the bone-specific protein osteocalcin. The osteocalcin gene promoter has become a model for understanding how genes are regulated, specifically in osteoblasts. One model for cell-specific regulation suggests that osteoblast-expressed genes are regulated through common promoter sequences which bind osteoblast-specific transcriptional activators. The phenotype suppression model suggests osteoblast-specific promoters are switched off through the action of the common transcriptional activator AP1. We previously demonstrated that a short sequence element (OSCARE-2) in the osteocalcin promoter was homologous to a repressive element in the collagen type 1 (alpha 1) promoters. In this paper we use electrophoretic mobility shift (EMS) assays to examine DNA-protein interactions in the OSCARE-2 sequence. In EMS assays, OSCARE-2 binds a complex of proteins, including AP1. This supports the role of AP1 sites in contributing to the regulation of the osteocalcin promoter. Exogenous c-JUN protein bound to OSCARE-2 and increasing c-JUN incubated with nuclear extract amounts caused a progressive increase in a higher-molecular-weight complex, consistent with c-JUN involvement in protein-protein as well as DNA-protein interactions. Anti-c-FOS antibody was capable of supershifting OSCARE-2 DNA-protein complexes produced using osteoblast-like cell nuclear extracts. In addition, EMS assays of nuclear proteins from osteoblast-like cells indicated that 1,25 (OH)2D3-inducible proteins are bound to OSCARE-2. Osteocalcin promoter constructs showed that OSCARE-2 contributed to the 1,25 (OH)2D3 response, albeit in a minor way. These data support the role of AP1 protein as a regulator of osteoblast-specific gene expression during osteoblast development.

Base Sequence↗
PubMed1990

Promotion of calvarial cell osteogenesis by endothelial cells.

Bone development and remodeling are associated with changes in the pattern of vascularization. Here we show that endothelial cells isolated from rat liver or bovine aorta can greatly enhance bone formation when implanted in diffusion chambers with rat fetal calvarial cells. The latter cells are unable to form bone when implanted alone at low initial cell density. The amount of mineralization measured by calcium deposition was 70 times higher in chambers containing calvarial cells mixed with endothelial cells from isologous liver or bovine aorta than in chambers containing endothelial or calvarial cells alone. Alkaline phosphatase activity was increased 20-fold. Calvarial cells in the presence of demineralized bone matrix powder did not form bone when implanted under similar conditions. Endothelial cells implanted alone seemed to enhance neovascularization around the Millipore diffusion chambers.

Alkaline Phosphatase↗
PubMed1995

Osteocalcin cluster: implications for functional studies.

Osteocalcin is a skeletal member of the family of extracellular mineral binding Gla protein. Osteocalcin is synthesized only by the osteoblast and it is secreted into the bone matrix at the time of bone mineralization. The mineral binding properties of osteocalcin as well as its spatial and temporal pattern of expression suggest that it plays a role during bone mineralization, however until now its biological function is unclear. To understand osteocalcin function during skeletogenesis we mutated the two osteocalcin genes by homologous recombination in embryonic stem (ES) cells. Eight targeted clones were identified by Southern analysis using external probes. One of these clones contributed to the germ line of mouse chimera. Interbreeding of heterozygotes is currently in progress. Mutant mice will be useful to understand osteocalcin function in vivo.

Animals↗
PubMed2000

[Bone tumors and tumor-like lesions of the jaw. Findings from the Basel DOSAK reference registry].

Within this chapter, tumors which produce bone matrix (osteoid) or other bone-forming tissues, like cartilage, connective tissue, and cementum or originate within the bone marrow are described. In addition, those lesions are mentioned which, according to the WHO, are typical for the jawbones and are therefore included within the 1992 WHO classification of odontogenic tumors. Vascular and epithelial tumors which may also be observed in the jaws are not described. All the lesions and tumors reported have in common a more or less similar clinical and radiological appearance. A precise diagnosis, therefore, can only be established by a sufficient biopsy which should be seen by a pathologist with experience in this field. In addition to clinical, radiological, and histological appearances, therapeutic necessities are described. The data from recent publications are taken into account. This presentation is based on the data collected within the reference registry of the German-Austrian-Swiss Study Group of Tumors of the Face and Jaws (DOSAK) in Basel.

Bone Neoplasms↗
PubMed1997

An electron microscopic study on the process of acid demineralization of cortical bone.

Demineralization has been shown to foster osteoinductive properties of cortical bone grafts, yet little is known about the process of demineralization and how to control it. The purpose of this study was to investigate the process of cortical bone demineralization by using scanning electron microscopy to evaluate how hydrochloric acid demineralizes cortical bone. Results showed that in the demineralization of diaphyseal cortical bone specimens using hydrochloric acid, a uniformly thick circumferential band of demineralized bone matrix surrounds an inner undecalcified bone core as the process of demineralization occurs. The interface between the demineralized and mineralized section of the bone specimens was extremely sharp. This interface between demineralized and undemineralized bone was noted to advance as a reaction front with increasing demineralization which resulted in continuous shrinkage of the inner cortical bone core. This study suggests that cortical bone demineralization can be best described using an advancing reaction front theory, and this explanation can be used for implementation of the concept of controlled demineralization.

Animals↗
PubMed1999

In situ hybridization with polymerase chain reaction-derived single-stranded DNA probe and S1 nuclease.

A rapid and simplified protocol for in situ hybridization (ISH) with polymerase chain reaction (PCR)-derived single-stranded DNA probes and S1 nuclease revealed transcripts of bone matrix proteins on decalcified skeletal bone specimens. Mouse bone tissue was fixed with 4% paraformaldehyde, decalcified with 20% EDTA, and embedded in paraffin. Each pair of primers for reverse transcriptase -PCR was designed to amplify a 280-bp DNA fragment from the coding region of the mature protein of mouse osteonectin (ON) and a 320-bp fragment from the coding region of mouse osteopontin (OP). Initial PCR products were eluted, purified, and reamplified by unidirectional PCR in the presence of the digoxigenin (DIG)-labeled dUTP. ISH was carried out by proteinase K treatment, hybridization, and washing. The unhybridized single-stranded DNA probe was selectively removed by S1 nuclease treatment. Hybridized probes were visualized with the alkaline phosphatase-conjugated anti-DIG antibody. The transcripts of ON and OP were clearly detected on the thin sections of the decalcified bone. Because this protocol does not require cloning or in vitro transcription, reliable and stable ISH can be done in an ordinary laboratory equipped with a thermal cycler.

Animals↗
PubMed2006

Role of DC-STAMP in cellular fusion of osteoclasts and macrophage giant cells.

Osteoclasts are the only cells that can resorb bone matrix physiologically and maintain the bone content. Osteoclasts are derived from macrophage/monocyte lineage cells; stimulation by macrophage colony stimulating factor and receptor activator of NFkappaB ligand induces osteoclastogenesis. During osteoclastogenesis, preosteoclasts fuse to form multinuclear mature osteoclasts. Cellular fusion is a unique phenomenon and enables fertilization, myotube formation, and efficient bone resorption in vertebrates. To date, several molecules have been reported to be fusion related in osteoclasts, namely CD44, CD47, ADAM12, MCP-1, and CD9, although the molecules which regulate osteoclast cellular fusion remain unclear. Here, we show that the seven-transmembrane-region receptor dendritic cell-specific transmembrane protein (DC-STAMP) is required for cell-cell fusion of osteoclasts and foreign body giant cells.

Adaptor Proteins, Signal Transducing↗
PubMed1990

Autoradiographic localization of osteogenin binding sites in cartilage and bone during rat embryonic development.

Osteogenin, a novel bone differentiation factor isolated from bone, has been recently purified and the amino acid sequence determined. Osteogenin in conjunction with a collagenous bone matrix substratum induces cartilage and bone formation in vivo. In order to understand the developmental role of osteogenin during cartilage and bone morphogenesis we examined the binding and distribution of iodinated osteogenin in developing rat embryos. Whole embryo tissue sections were made from 11, 12, 13, 15, 18, and 20 day fetuses. The specific binding of osteogenin at different stages of rat embryonic development was determined by autoradiography. Maximal binding was observed in mesodermal tissues such as cartilage, bone, perichondrium, and periosteum. During Days 11-15, peak binding was localized to perichondrium during limb and vertebral morphogenesis. By Day 18 periosteum exhibited the highest concentration of autoradiographic grains. Osteogenin was also localized in developing membranous bones of the calvarium and other craniofacial bones. Considerably less binding was observed, in decreasing order, in muscle, liver, spleen, skin, brain, heart, kidney, and intestine. The observed maximal binding during skeletal morphogenesis implies a developmental role for osteogenin.

Animals↗
PubMed2005

Innate immunity in human bone.

Bone has exceptional regenerative properties. Oral bone appears to be particularly resistant to infection despite exposure to oral flora, even in circumstances such as oral surgery where the thin mucosal layer covering the bone is disrupted. The goal of this study was to determine whether the innate immune system of antimicrobial peptides exists inside bone. Biopsies of non-infected and chronically infected mandibular bone were harvested from patients during maxillofacial surgical procedures. Bone biopsies from the iliac crest and fibula served as controls. Immunohistochemical staining was performed, directed against the human beta-defensin antimicrobial peptides (hBD) -1, -2 and -3. In addition, cultures of osteoblast-like cells were examined for the presence of each of the three beta-defensins and their mRNA transcripts. All three human beta-defensins were detected within the mineralized bone matrix of chronically infected mandibular bone in the vicinity of the endosteum and osteocytes. hBD-1, -2 and -3 were also found in the cytoplasm of osteocytes. Expression of all three beta-defensins was detected in each of the non-infected bone types including the controls, however, to a lesser degree than that found in the chronically infected mandibular bone. This may reflect upregulation of antimicrobial peptide expression in the presence of chronic infection. Cultures of non-infected osteoblast-like cells were found to express mRNA for each of hBD-1, -2 and -3. Immunohistochemical staining of the cultures was positive for hBD-1 and -2, but not for hBD-3. We provide the first evidence of a previously unrecognized innate immunological function of bone through the demonstration of the presence of the human beta-defensins hBD-1, -2 and -3 in bone.

Alkaline Phosphatase↗